The interaction of a beta-adrenergic agonist with the beta-adrenoceptor results in the activation of the enzyme adenylate cyclase. Although the molecular mechanism of adenylate cyclase activation is unknown, the regulation of enzyme activity appears to involve several distinct regulatory sites including the catalytic subunit of the enzyme, the hormone receptor and possibly two guanine nucleotide (GTP) binding proteins one of which may have GTPase activity. The proposed study concerns the regulation and characterization of the GTP binding protein (N-site) that is coupled to adenylate cyclase and requires GTP to be present for hormone activation of the enzyme to occur. Using a recently developed (3H) 5'-guanylyl-imidodiphosphate (Gpp(NH)p) binding assay, regulation of the N-site will be determined in the lung during normal growth and in animals treated with thyroid hormone, beta-adrenergic agonists, glucocorticoids, a catecholamine depleting agent (resperine) and chemical denervation (6-hydroxydopamine). The density of beta-adrenoceptors during normal growth of the lung and in drug treated animals will also be determined and the ratio of N-sites to beta-adrenoceptors calculated (normal tissue has a 1:1 ratio). If the ratio changes, Ka values for adenylate cyclase activation by beta-agonist and GTP will be determined. Characterization of the N-site will include the kinetics of (3H)GPP(NH)p binding and how membrane perturbants (phospholipases, proteases) and protein modifying agents (thiol reagents) effect this protein. After solubilization of the N-site by detergents, attempts will be made to purify the N-site by affinity chromatography and/or conventional techniques. The partially purified protein will be characterized with respect to binding properties, molecular weight, subunit binding site and, if present, GTPase activity. The proposed study may contribute to our understanding of the nature of a critical regulatory unit (N-site) involved in the control of airways resistance and possibly surfactant secretion. The possibility exists for a new class of drugs working through the N-site to control lung function.